Separating and concentrating cells from bulk solutions for analysis is a nontrivial task in life science research, diagnostics, and industrial processing. As such, various approaches based on physical or biochemical properties of cells have been developed to improve separation efficiency. Biochemical moieties on cell surfaces are most commonly used to distinguish cell populations, in which a specific receptor or protein is targeted with a recognition element (e.g., antibody, aptamer, ligand) to yield a fluorescent or magnetic label enabling downstream sorting.
The most widely used cell sorting techniques of this nature are fluorescence-activated cell sorting (FACS) and magnetic activated cell sorting (MACS). In FACS, multiple cell populations can be separated from heterogeneous mixtures based on the quantity of fluorophore associated with the cell. Though effective, the FACS process is performed serially and each cell is analyzed individually, increasing processing times for large sample volumes. Comparatively, magnetic based approaches are advantageous due to their simplicity and robustness, not requiring sophisticated fluid handling. These approaches are also able to operate on minimal cell quantities and/or process larger volumes more rapidly. However, magnetic separation approaches remain less quantitative than FACS, which can gate on the relative quantity of a biomarker. This lack of quantification from traditional MACS is due to the fact that these approaches cannot discriminate effectively based on the number of bound magnetic particles. Additionally, some magnetic particles available today are not tightly controlled in size or magnetic content, further exacerbating efforts for quantification of biomarker levels as correlated to number of bound particles, emphasizing the need for techniques to purify particles based on magnetic content.
Several microfluidics approaches have been developed to quantitatively separate cells based on bound or internalized magnetic content. In general, these techniques involve generating a magnetophoretic force orthogonal to a fluid flow direction, inducing cell deflection across streamlines and separation into different outlets depending on magnetic content. However, these “kinetic” based separations require precise tuning of flow rate, fluidic resistance, and magnetic field positioning. Additionally, many of these systems have low throughput as they rely on weaker bulk magnetic field gradients. Finally, the output from flow-through based systems often yields diluted solutions which may require additional concentration steps and is particularly challenging for isolating and locating rare cell types.
Magnetic ratcheting has the potential to achieve quantitative magnetic separations to both purify magnetic particle populations and separate cells based on bound number of particles. In magnetic ratcheting, arrays of magnetic micro-pillars combined with a directionally cycled magnetic field create dynamic potential energy wells that trap and manipulate magnetic particles in a magnetic-content and particle-size dependent manner. However, previous ratcheting platforms have utilized thin film magnetic structures (height≤200 nm), which have minimal force capacities on the order of 10 pN, due to the low aspect ratio of the structures used. To compensate, larger particles are used (˜3-10 μm) to maximize the force envelope. However, larger particles have reduced magnetic labeling efficiency for cell separations due to slow diffusive motions. This slow diffusive motion results in inefficient binding of large particles to cell surface targets and the large increment in magnetic content per bound particle makes it difficult to relate bead binding to target expression levels. The use of smaller magnetic particles is necessary to increase labeling efficiency as well as provide a sensitive metric to relate bound particle numbers with cell surface expression, but is not practically compatible with current ratcheting technology. Additionally, previous ratcheting platforms rely on velocity differences between particles to achieve magnetic based separation. Again this is a “kinetic” separation requiring initial sample concentration prior to process initiation, and time dependent collection functions. These challenges have limited use as a quantitative sorting tool. Ideally, an equilibrium separation could achieve reduced dependence on initial and final conditions of a sample yielding a more robust and quantitative separation.